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In order to characterize the development of polyphenic traits in the wings, flight muscles and gonads of the soapberry bug, Jadera haematoloma, we sequenced transcriptomes from a multidimensional matrix of life stage, tissue, sex, food regime and host population.

Sampling design

Transcriptome studies proceeded through three phases. Initially, we sampled whole bodies from 12 individuals, including three biological replicates of each sex and morph combination from the Plantation Key, FL population (phase 1). Next we sampled dorsal thorax and gonads from three individuals of each sex and morph combination from the Plantation Key, FL and Aurora, CO populations (phase 2). Samples from these phases were prepared as full-length 150-bp paired-end Illumina libraries and used to assembly a reference transcriptome.

The final phase utilized 3’-tag sequencing with the exclusive goal of quantifying gene expression. The sampling design at this phase included two stages (fifth instars and nascent adults), two tissues (dorsal thorax and gonads), two sexes, long- and short-wing adult morphs, high and low food regimes, four populations (collected from Key Largo, FL, Plantation Key, FL, Aurora, CO, and Frederick, MD), and three biological replicates of each unique combination of the preceding factors. Thus the full matrix contained 288 samples. Seven samples were lost or did not pass library quality controls, resulting in 281 samples in the analysis.

Nucleic acid extractions

Individuals were collected from laboratory cultures, anesthetized using CO2 exposure, photographed, and flash frozen in liquid nitrogen. Samples were stored at -80˚C until further processing. The thorax was removed from head and abdomen with a clean scalpel blade, and the dorsal thorax was separated above the legs while tissue was still frozen. Gonads were then dissected from the abdomen of adults. For juveniles, the entire abdomen was included. Nucleic acid extractions used the Invitrogen PureLink RNA extraction kit.

Sequencing and assembly of a reference transcriptome

Samples used for transcriptome assembly (phases 1 and 2) were delivered on dry ice to Beckman Coulter Genomics (Danvers, Massachusetts) for poly-A selection, preparation of 125 bp paired-end TruSeq libraries, and sequencing using Illumina HiSeq. Libraries from phases 1 and 2 were sequenced separately. Reads from all samples were trimmed using Trimmomatic version 0.33 (Bolger et al. 2014) with the parameters ILLUMINACLIP:/export/local/src/Trimmomatic-0.33/adapters/TruSeq3-SE.fa:2:30:10 HEADCROP:12 LEADING:3 TRAILING:3 SLIDINGWINDOW:3:15 MINLEN:50. The resulting reads were assembled into transcripts by Trinity version 2.0.6 (Grabherr et al. 2011). Before annotation, potentially redundant transcripts with greater than 90% identity were collapsed using CD-HIT-EST version 4.6 (Li & Godzik 2006).

cd /research/drangeli/phase2_BCG_RNAseq/Jhae_GU_trinity_assembly/
cd-hit-est -i GU.Trinity.fa -o GU.Trinity.c90.fa -c 0.90 -n 10 -d 0 -M 64000 -T 12

This took about 3.5 days on node 26.

TransDecoder identified coding sequences. At each stage, BUSCO version 5.2.2 (Manni et al. 2021) was used to assess transcriptome completeness.

busco --in GU.Trinity.fa \
--lineage_dataset /research/drangeli/DB/busco.lineages/hemiptera_odb10 \
--out BUSCO.report.for.GU \
--augustus --augustus_species fly --mode transcriptome --cpu 24

Consolidation by cd-hist-est dramatically reduced the number of sequences flagged as duplicates (Table S1).

Table S1: Details of transcriptome datasets at each stage of processing.

dataset contigs BUSCO short summary
initial assembly 549,485 C:97.0%[S:54.2%,D:42.8%],F:1.3%,M:1.7%,n:2510
initial assembly CDS 85,937
90% consolidation 395,125 C:96.6%[S:77.7%,D:18.9%],F:1.4%,M:2.0%,n:2510
90% consolidation CDS 50,771 C:94.4%[S:77.8%,D:16.6%],F:2.2%,M:3.4%,n:2510

Annotation of the transcriptome

Transcript annotation was performed by EnTAP (Hart et al. 2019) using the NCBI invertebrate RefSeq dataset, SwissProt and Trembl databases. EnTAP assigns sequence names and GO terms based on similarity.

EnTAP --runP --threads 24 \
-i /research/drangeli/phase2_BCG_RNAseq/Jhae_GU_trinity_assembly/GU.Trinity.c90.fa \
-d /export/groups/drangeli/entap_config/bin/invert.dmnd \
-d /export/groups/drangeli/entap_config/bin/uniprot_sprot.dmnd \
-d /export/groups/drangeli/entap_config/bin/uniprot_trembl.dmnd \
--ini /export/groups/drangeli/Jhae.genome/entap_config.ini

The J. haematoloma sequences were matched to other Hemiptera, primarily Halyomorpha halys, Cimex lectularius and Nilaparavata lugens for the RefSeq comparisons and Oncopeltus, Lygus and Riptortus from TrEMBL. The contaminant sequence species are also as expected, coming from bacteria and yeasts. Only 12 potential contaminant sequences had homology to humans.

Sequencing for gene expression quantification

For gene expression analysis, RNA samples were shipped overnight on dry ice to the DNA Technologies Core at the University of California at Davis. Libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Greenland, New Hampshire, USA) in the three batches for sequencing in separate Illumina HiSeq lanes. Batch 3 was sequenced twice and reads counts for each run were added together for each sample after filtering.

These batches were sequenced in three lanes yielding 115.9×109 bp. These data

Short read sequences were inspected for quality using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), before and after trimmed using Trimmomatic version 0.33, as in the example command below.

java -jar trimmomatic-0.33.jar SE \
raw.data/A1-d25-2_S161_L006_R1_001.fastq \
filtered.reads/A1-d25-2_S161_L006_R1_001.trimmed.fastq \
ILLUMINACLIP:/export/local/src/Trimmomatic-0.33/adapters/TruSeq3-SE.fa:2:30:10 \
HEADCROP:16 LEADING:3 TRAILING:3 SLIDINGWINDOW:3:15 MINLEN:79

A simple bash script was used to apply trimming to all read files and save the output in a separate folder.FastQC inspection after trimming revealed notable improvements.

Table S2: Read numbers before and after quality filtering.

batch raw reads filtered reads passing filter
1 341,370,316 323,188,378 94.7%
2 321,564,969 313,590,249 97.5%
3 484,588,962 419,931,564 86.7%
overall 1,147,524,247 1,056,710,191 92.1%

Mapping 3seq reads to the transcriptome

Kallisto was used to quantify the abundance of transcripts from 3seq data using a pseudo-alignment method (Bray et al. 2016). First, a Kallisto index was created from the assembled transcripts, collapsed by cd-hit-est.

We chose to use this target, rather than the TransDecoder CDS files, because if there are fragmentary or non-coding RNAs that were sequenced, then some reads will correspond to those RNAs too.

cd /research/drangeli/phase2_BCG_RNAseq/Jhae_GU_trinity_assembly/
kallisto index --index=GU.Trinity.c90.kallisto.index GU.Trinity.c90.fa

In order to run Kallisto on single-end sequence, the program requires the mean and standard deviation of read lengths. The total number of reads was found using bash commands.

cd /export/groups/drangeli/morphDE/davis.3seq/filtered.reads

gunzip -c 180824/*.fastq.gz 190917/*.fastq.gz 200611/*.fastq.gz 200701/*.fastq.gz \
| sed -n -e '2~4p' \
| awk '{ print length($0); }' > read.lengths.txt

wc -l read.lengths.txt

Total read number was 1,149,235,725.

Mean and standard deviation were calculated using awk.

cat read.lengths.txt | awk '{for(i=1;i<=NF;i++) {sum[i] += $i; sumsq[i] += ($i)^2}} END {for (i=1;i<=NF;i++) { printf "%f %f \n", sum[i]/NR, sqrt((sumsq[i]-sum[i]^2/NR)/NR)} }'

A bash script was used to run kallisto quant for each sample and to rename the output files with the sample names.

The result is a folder kallisto.vs.JhaeGUtx that contains .abundance.tsv files for each sample, listing the counts for each transcript.

The resulting output files were then merged into a single comma-separated values (CSV) file with columns for each sample.

This file kallisto.counts.csv is 309 Mb, which is too large to post to GitHub.

Differential gene expression analysis

Gene expression differences were examined using the Bioconductor package DESeq2 version 1.32.0 (Love et al. 2014) in R version 4.1.1 (R Core Team 2021). The function DESeq fits a generalized linear model where counts are modeled using a negative binomial distribution with a gene-specific dispersion parameter. Means were estimated using approximate posterior estimation for generalized linear models (Zhu et al. 2018). We extracted log2 fold-change and p-values corrected by independent hypothesis weighing (Ignatiadis et al. 2016) and applied a critical threshold of 0.05. Contrasts were made for sex, wing morph and food regime, after accounting for batch effects (multiple sequencing runs). Data were analyzed separately for each stage (fifth instar juveniles and nascent adults) and for each tissue (thorax and gonads). Differential gene expression was visualized by customized plots generated using ggplot2. Variance stabilizing transformation, using the function vst, was applied before ordination using principle component analysis.

For more details see the R script analysis.dge.R

Batch effects were introduced by the need to sequence samples in multiple lanes (Figure S1).

Figure S1. Principle components of uncorrected counts, color-coded by biological factors (A) and sequencing batch (B).

DESeq2 allows variance from confounding factors, such as sequencing batch, to be accounted for through generalized linear modeling. The normalized counts that result from the model then reflect biologically meaningful variance (Figure S2).

Figure S2. Principle components of transcript counts for each sample, following normalization for sequencing run (batch). Samples are color-coded by biological origin.

More complex models can be applied to examine specific contrasts of biological interest. These models partition the variance in gene expression sequentially to factors in the model. For more complex models, less power exists to detect differences, but it allows for the influence of multiple factors to be disentangle from one another.

Expression differences by sex

Sex is often an important factor effecting gene expression. While it is not our focus of study, examining our dataset for differences between females and males in each tissue is a useful confirmation of these expectations (Figure S3. Moreover, sexually dimorphic gene expression has not previously been described in J. haematoloma. Genes were differentially expressed at both sampled stages and in both sampled tissues. Adults had a larger number of sexually dimorphic DEGs (84% in gonad; 29% in thorax) compared to juveniles (6.1% in gonads, 0.18% in thorax). The gonads also had more sexually dimorphic gene expression than did the thorax. Within the gonads, males had more significantly biased genes than did females, at both stages (Figure S3C-D). Several genes with roles in somatic sex determination were among these, including doublesex (dsx) and transformer [ref]. The histone acetyltransferase encoded by mof is known to increase expression on the X chromosome in D. melanogaster males (Smith et al. 2000), although the mof ortholog appears up-regulated in the J. haematoloma female thorax (Fig S3B). In the adult gonad contrast, upd is significantly biased towards male expression. In D. melanogaster testis, upd is expressed by hub cells promote maintenance of spermatogenic stem cells (reviewed by Matunis et al 2012).

Figure S3. Volcano plots, showing log2 fold-change (LFC) and -log10 FDR-adjusted p-values for contrasts by sex in the juvenile thorax (A), adult thorax (B), juvenile gonads (C) and adult gonads (D). Genes are highlighted in red if they have an absolute LFC values > 1 (two-fold difference) and an FDR-adjusted p-value < 0.05 (panels A) or 10-3 (panels B-D). Highlighted transcript are annotated where possible.

Expression differences by morph

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Figure S4. Volcano plots, showing log2 fold-change (LFC) and -log10 FDR-adjusted p-values for contrasts by morph in the adult thorax (A), adult gonads (B), adult ovaries (C) and adult testes (D). Genes are highlighted in red if they have an absolute LFC values > 1 (two-fold difference) and an FDR-adjusted p-value < 10-3 (panels A) or 0.05 (panels B-D). Highlighted transcript are annotated where possible.

Expression differences by stage

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Figure S5. Volcano plots, showing log2 fold-change (LFC) and -log10 FDR-adjusted p-values for contrasts by stage in the thorax (A), ovaries (B), and testes (C). Genes are highlighted in red if they have an absolute LFC values > 1 (two-fold difference) and an FDR-adjusted p-value < 10-3. Highlighted transcript are annotated where possible.

Expression differences by food regime

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Figure S5. Volcano plots, showing log2 fold-change (LFC) and -log10 FDR-adjusted p-values for contrasts by food regime in the juvenile thorax (A), adult thorax (B), juvenile gonads (C), and adult gonads (D). Genes are highlighted in red if they have an absolute LFC values > 1 (two-fold difference) and an FDR-adjusted p-value < 0.05. Highlighted transcript are annotated where possible.

Enrichment analysis

Enrichment analyses were performed based on annotations with terms from the GO and KEGG ontologies, as well as EggNOG protein domains, applied to transcripts by EnTAP. We also tested for enrichment of xenic or contaminant transcripts among those that were differentially expressed, and neither of these categories was significantly over-represented. For each term, p-values from the hypergeometric test, implemented in the base R function phyper, were adjusted for false discovery rates (FDR).

For more details on the enrichment analysis see the R script enrichment.R

Table S3: Terms from GO and KEGG ontologies and EggNOG protein domains over-represented in differentially expressed genes in each dataset and contrast.

tissue stage contrast ontology enriched terms top terms
thorax - by stage GO 1670 GO:0005622(L=3) intracellular anatomical structure, 1.09×10-104; GO:0071704(L=2) organic substance metabolic process, 4.2×10-97; GO:0043229(L=3) intracellular organelle, 2.40×10-91; GO:0044238(L=2) primary metabolic process, 7.21×10-90; GO:0044237(L=2) cellular metabolic process, 1.21×10-82; GO:0005737(L=4) cytoplasm, 1.87×10^-76; GO:0043227(L=2) membrane-bounded organelle, 8.45×10-66; GO:0043231(L=4) intracellular membrane-bounded organelle, 8.95×10-66; GO:0048856(L=2) anatomical structure development, 1.71×10-57; GO:0007275(L=3) multicellular organism development, 4.9×10-53
thorax - by stage KEGG 68 map01100 Metabolic pathways, 5.042×10-44; map05012 Parkinson disease, 3.85×10-14; map01110 Biosynthesis of secondary metabolites, 2.84×10-12; map05010 Alzheimer disease, 2.12×10-8; map00500 Starch and sucrose metabolism, 4.38×10-7; map04145 Phagosome, 5.02×10-7; map00982 Drug metabolism - cytochrome P450, 2.19×10-6; map00980 Metabolism of xenobiotics by cytochrome P450, 3.82×10-6; map00280 Valine, leucine and isoleucine degradation, 7.08×10-6; map03410 Base excision repair, 1.34×10-5
thorax - by stage EggNOG 87 TRANS, 1.71×10-120; SIGNAL, 1.21×10-98; COIL, 1.78×10-53; Insect cuticle protein, 9.19×10-21; Major facilitator superfamily, 4.55×10-08; Chitin binding peritrophin-A domain, 3.66×10-07; RNA recognition motif, 3.66×10-07; Chitin-binding domain type 2, 1.08×10-06; Sugar transporter, 2.52×10-06; A1 Propeptide, 5.70×10-06
testes - by stage GO 0
testes - by stage KEGG 0
testes - by stage EggNOG 0
ovaries - by stage GO 9 GO:0006260(L=5) DNA replication, 1.10×10-9; GO:0034061(L=5) DNA polymerase activity, 1.19×10-8; GO:0003964(L=6) RNA-directed DNA polymerase activity, 5.30×10-7; GO:0006278(L=7) RNA-dependent DNA biosynthetic process, 5.30×10-7; GO:0006259(L=4) DNA metabolic process, 1.70×10-6; GO:0016779(L=4) nucleotidyltransferase activity, 0.000765; GO:0015074(L=5) DNA integration, 0.007542; GO:000452(L=8) RNA-DNA hybrid ribonuclease activity, 0.0124; GO:0005319(L=3) lipid transporter activity, 0.0414; GO:0070001(L=5) aspartic-type peptidase activity, 0.0507
ovaries - by stage KEGG 0
ovaries - by stage EggNOG 5 reverse transcriptase, 1.58×10-8; Rnase H, 0.00235; Endonuclease/Exonuclease/phosphatase family, 0.00240; Integrase, 0.00240; ZnF, 0.0339
thorax L5 by sex GO 0
thorax L5 by sex KEGG 0
thorax L5 by sex EggNOG 15 dsx/mab-3 DNA-binding domain, 9.13x10-7; Complementary sex determiner-like SR domain, 0.0180; proline-rich protein BAT2 N-terminal domain, 0.0180; CD34/Podocalyxin family, 0.0180; DFDF domain, 0.0180; Sex determination protein N terminal domain, 0.0180; Barentsz/CASC3 domain, 0.0215; Meckelin, 0.0215; CDC48, 0.0298
thorax adult by sex GO 267 GO:0022626(L=5) cytosolic ribosome, 2.98×10^-41; GO:0006412(L=5) translation, 1.07×10-29; GO:0003723(L=4) RNA binding, 1.08×10-27; GO:0034645(L=4) cellular macromolecule biosynthetic process, 4.73×10-27; GO:0009059(L=4) macromolecule biosynthetic process, 1.16×10-26; GO:0000022(L=5) mitotic spindle elongation, 4.66×10-26; GO:0051231(L=4) spindle elongation, 3.81×10-25; GO:0044391(L=5) ribosomal subunit, 5.19×10-25; GO:0022625(L=6) cytosolic large ribosomal subunit, 9.80×10-24; GO:0044249(L=3) cellular biosynthetic process, 3.9×10-23
thorax adult by sex KEGG 3 map04974 Protein digestion and absorption, 6.71x10-6; map04972 Pancreatic secretion, 1.04x10-4; map00513 Various types of N-glycan biosynthesis,7.60x10-3
thorax adult by sex EggNOG 26 Reverse transcriptase, 2.11x10-9; Endonuclease/Exonuclease/phosphatase family, 1.38x10-8; Rnase H, 7.70x10-7; ZnF C2HC, 5.78x10-6; Lipoprotein N-terminal domain, 6.48x10-5; Vitellogenin-like N-terminal domain, 6.48x10-5; DUF1943, 2.54x10-4; Co/Zn superoxide dismutase, 8.63x10-4; CUB domain, 1.77x10-3; Trypsin, 3.17x10^-3
gonad L5 by sex GO 1299 GO:0005622(L=3) intracellular anatomical structure, 1.40×10-94; GO:0043229(L=3) intracellular organelle, 7.32×10-84; GO:0043231(L=4) intracellular membrane-bounded organelle, 1.47×10-64; GO:0043227(L=2) membrane-bounded organelle, 5.40×10-64; GO:0005737(L=4) cytoplasm, 2.40×10-55; GO:0044237(L=2) cellular metabolic process, 7.05×10-55; GO:0044238(L=2) primary metabolic process, 1.08×10-52; GO:0016043(L=2) cellular component organization, 3.73×1-51; GO:0071704(L=2) organic substance metabolic process, 6.15×10-50; GO:0043228(L=2) non-membrane-bounded organelle, 1.66×10-44
gonad L5 by sex KEGG 31 map01100 Metabolic pathways, 4.58×10-9; map05012 Parkinson disease, 1.22×10-7; map00710 Carbon fixation in photosynthetic organisms, 1.83×10-6; map05010 Alzheimer disease, 3.77×10-6; map00051 Fructose and mannose metabolism, 9.74×10-5; map01110 Biosynthesis of secondary metabolites, 1.04×10-4; map00030 Pentose phosphate pathway, 9.86×10-4; map04340 Hedgehog signaling pathway, 9.86×10-4; map05152 Tuberculosis, 9.86×10-4; map05110 Vibrio cholerae infection, 1.13×10-3
gonad L5 by sex EggNOG 46 COIL, 1.62x10-69; S/T kinase, 5.50x10-9; TRANS, 3.37x10-8; WD40 domain, 3.31x10-7; RNA recognition motif, 5.60x10-7; protein kinase, 1.14x10-6; Protein tyrosine kinase, 1.14x10-6; RNA recognition motif, 1.28x10-6; M17 cytosol aminopeptidase, 2.15x10-5; Leucin-rich repeats, 4.30x10-5
gonad adult by sex GO 0
gonad adult by sex KEGG 0
gonad adult by sex EggNOG 0
thorax adult by morph GO 1251 GO:0005737(L=4) cytoplasm, 1.67×10-124; GO:0005622(L=3) intracellular anatomical structure, 1.68×10-118; GO:0005739(L=5) mitochondrion, 1.4×10-100; GO:0043229(L=3) intracellular organelle, 4.93×10-98; GO:0043227(L=2) membrane-bounded organelle, 4.05×10-87; GO:0043231(L=4) intracellular membrane-bounded organelle, 7.23×10-87; GO:0044237(L=2) cellular metabolic process, 1.30×10-68; GO:0071704(L=2) organic substance metabolic process, 2.37×10-56; GO:0044238(L=2) primary metabolic process, 2.97×10-56; GO:0005759(L=6) mitochondrial matrix, 6.3×10-50
thorax adult by morph KEGG 16 map01100 Metabolic pathways, 5.266×10-32; map05012 Parkinson disease, 3.35×10-26; map05010 Alzheimer disease, 5.08×10-19; map01110 Biosynthesis of secondary metabolites, 7.45×10-13; map04974 Protein digestion and absorption, 7.02×10-6; map00640 Propanoate metabolism, 1.16×10-3; map00513 Various types of N-glycan biosynthesis, 1.21×10-3; map01120 Microbial metabolism in diverse environments, 2.22×10-3; map04260 Cardiac muscle contraction, 2.92×10-3; map00020 Citrate cycle (TCA cycle) 4.59×10-3
thorax adult by morph EggNOG 48 COIL, 4.72×10-41; TRANS, 5.43×10-40; SIGNAL, 4.54×10-19; Immunoglobulin I-set domain, 2.84×10-5; Immunoglobulin C-2 Type, 3.53×10-5; Immunoglobulin V-set domain, 3.77×10-5; CUB domain, 4.06×10-5; Immunoglobulin domain, 1.15×10-4; EF-hand calcium binding motif, 9.41×10-4; Allatostatin, 2.30×10-3
testes adult by morph GO 0 GO:0003401(L=5) axis elongation, 0.07783; GO:000340(L=9) planar cell polarity pathway involved in axis elongation, 0.0778; GO:0005927(L=4) muscle tendon junction, 0.0778; GO:0018108(L=7) peptidyl-tyrosine phosphorylation, 0.0778; GO:0018212(L=7) peptidyl-tyrosine modification, 0.0778; GO:0060071(L=8) Wnt signaling pathway, planar cell polarity pathway, 0.0778; GO:0001764(L=7) neuron migration, 0.084; GO:0003382(L=6) epithelial cell morphogenesis, 0.084; GO:0035567(L=6) non-canonical Wnt signaling pathway, 0.084; GO:0090175(L=6) regulation of establishment of planar polarity, 0.084
testes adult by morph KEGG 7 map04110 Cell cycle, 0.0105; map04360 Axon guidance, 0.0105; map05130 Pathogenic Escherichia coli infection, 0.0105; map05131 Shigellosis, 0.0105; map05220 Chronic myeloid leukemia, 0.0105; map04722 Neurotrophin signaling, 0.0197; map05200 Pathways in cancer, 0.0327
testes adult by morph EggNOG 7 Mic1, 0.00428; F-actin binding domain, 0.00535; Src homology 2 domain, 0.0224; Tyrosine kinase, 0.0224; Src homology 3 domain, 0.0372
ovaries adult by morph GO 55 GO:0007552(L=4) metamorphosis, 0.008697; GO:0001540(L=4) amyloid-beta binding, 0.01583; GO:0004857(L=3) enzyme inhibitor activity, 0.01583; GO:0005540(L=4) hyaluronic acid binding, 0.01583; GO:0005606(L=6) laminin-1 complex, 0.01583; GO:0005874(L=6) microtubule, 0.01583; GO:0008234(L=5) cysteine-type peptidase activity, 0.01583; GO:0008773(L=6) [protein-PII] uridylyltransferase activity, 0.01583; GO:0009886(L=4) post-embryonic animal morphogenesis, 0.01583; GO:0010466(L=8) negative regulation of peptidase activity, 0.01583
ovaries adult by morph KEGG 28 map04020 Calcium signaling, 0.0233; map04062 Chemokine signaling, 0.0233; map04145 Phagosome, 0.0233; map04270 Vascular smooth muscle contraction, 0.0233; map04540 Gap junction, 0.0233; map04612 Antigen processing and presentation , 0.0233; map04713 Circadian entrainment , 0.0233; map04723 Retrograde endocannabinoid signaling, 0.0233; map04724 Glutamatergic synapse, 0.0233; map04725 Cholinergic synapse, 0.0233
ovaries adult by morph EggNOG 33 C8 domain, 0.0126; Adenylyl/guanylyl cyclase, 0.0126; Calcium-binding EGF-like domain, 0.0126; Androgen-induced protein 1-like domain, 0.0126; Cathepsin propeptide inhibitor-29 domain , 0.0126; Laminin EGF domain, 0.0126; Low-density lipoprotein receptor repeat class B, 0.0126; Low-density lipoprotein-receptor YWTD domain, 0.0126; Nuclear distribution protein
gonad adult by morph GO 370 GO:0001705(L=5) ectoderm formation, 0.009967; GO:0001712(L=6) ectodermal cell fate commitment, 0.009967; GO:0001715(L=7) ectodermal cell fate specification, 0.009967; GO:0001727(L=5) lipid kinase activity, 0.009967; GO:0004016(L=3) adenylate cyclase activity, 0.009967; GO:0005942(L=3) phosphatidylinositol 3-kinase complex, 0.009967; GO:0005943(L=5) phosphatidylinositol 3-kinase complex, class IA, 0.009967; GO:0006171(L=8) cAMP biosynthetic process, 0.009967; GO:0007154(L=2) cell communication, 0.009967; GO:0007165(L=3) signal transduction, 0.009967
gonad adult by morph KEGG 71 map04062 Chemokine signaling, 0.000229; map04725 Cholinergic synapse, 0.000229; map04914 Progesterone-mediated oocyte maturation, 0.000229; map04915 Estrogen signaling, 0.000319; map05166 Human T-cell leukemia virus 1 infection, 0.000348; map04020 Calcium signaling, 0.00627; map04070 Phosphatidylinositol signaling, 0.00627; map04150 mTOR signaling, 0.00627; map04210 Apoptosis, 0.00627; map04370 VEGF signaling, 0.00627; map04620 Toll-like receptor signaling, 0.00627; map04630 JAK-STAT signaling, 0.00627; map04911 Insulin secretion, 0.00627; map04913 Ovarian steroidogenesis, 0.00627
gonad adult by morph EggNOG 11 COIL, 0.00155; PI3-kinase C2 domain, 0.00155; PI3K p85-binding domain, 0.00155; PI3K ras-binding domain, 0.00155; PI3K accessory domain, 0.00155; Cyclin C, 0.00183; Guanylate cyclase catalytic domain, 0.00183; IPT domain, 0.00183; Phosphatidylinositol 3/4-kinase, 0.00183; PI3K catalytic domain, 0.00183
thorax L5 by food GO 2 GO:0016052(L=4) carbohydrate catabolic process, 0.03984; GO:0030246(L=2) carbohydrate binding, 0.03984; GO:0005975(L=3) carbohydrate metabolic process, 0.09147; GO:0009056(L=2) catabolic process, 0.1903; GO:1901575(L=3) organic substance catabolic process, 0.1903; GO:0044238(L=2) primary metabolic process, 0.4692; GO:0071704(L=2) organic substance metabolic process, 0.4692
thorax L5 by food KEGG 0
thorax L5 by food EggNOG 7 tetrapeptide XPTX repeats, 0.00144; catecholamine-binding domain, 0.00162; dopamine beta-monooxygenase N-terminal-like domain, 0.00162; SEA domain, 0.00162; EGF2, 0.00360; EGF-like, 0.00960; EGF, 0.00977
thorax adult by food GO 0
thorax adult by food KEGG 0
thorax adult by food EggNOG 5 DUF1943 domain, 0.01779; Lipoprotein N-terminal domain, 0.01779; Co/Zn superoxide dismutase, 0.01779; Vitellogenin N-terminal domain, 0.01779; von Willebrand factor type D domain, 0.01930
gonad L5 by food GO 0
gonad L5 by food KEGG 0
gonad L5 by food EggNOG 4 DAN domain, 0.00911; Peroxisome biogenesis factor 1, 0.00911; Trypsin-like serine protease, 0.0119; Trypsin, 0.0119
gonad adult by food GO 0
gonad adult by food KEGG 2 map04974 Protein digestion and absorption, 0.00304; map04972 Pancreatic secretion, 0.00420
gonad adult by food EggNOG 8 CUB domain, 0.0256; DMPK coiled coil, 0.0256; Trypsin-like serine protease, 0.0256; Trypsin, 0.0256; microtubule-associated protein 7 family, 0.0308; P21-Rho-binding domain, 0.0308; CNH domain, 0.0330; Sodium solute symporter, 0.0375

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